Patent application title: Use of fgf-21 and thiazolidinedione for treating type 2 diabetes

Abstract:

A method for treating type 2 diabetes and metabolic syndrome comprising
administering an effective amount of fibroblast growth factor 21 in
combination with a thiazolidinedione.

Claims:

1. A method for treating a mammal exhibiting type 2 diabetes or metabolic
syndrome comprising: administering to said mammal a therapeutically
effective amount of FGF-21 or an FGF-21 compound in combination with a
thiazolidinedione sufficient to achieve in said mammal at least one of
the following modifications: reduction in triglycerides, decrease in
insulin resistance, reduction of hyperinsulinemia, increase in glucose
tolerance, or reduction of hyperglycemia.

2. The method of claim 1 wherein said mammal is a human subject which
exhibits type 2 diabetes.

3. The method of claim 1 wherein said mammal is a human subject which
exhibits metabolic syndrome.

4. The method of claim 1 wherein said modification is a reduction of
hyperglycemia.

5. The method of claim 1 wherein said modification is a reduction in
triglycerides.

6. The method of claim 1 wherein said thiazolidinedione is selected from
the group consisting of rosiglitazone or pioglitazone.

7. The method of claim 6 wherein said thiazolidinedione is rosiglitazone.

8. The method of claim 6 wherein said thiazolidinedione is pioglitazone.

Description:

FIELD OF INVENTION

[0001]This invention relates to the use of fibroblast growth factor 21 in
combination with a thiazolidinedione for the treatment of mammals
suffering from non-insulin dependent Diabetes Mellitus (NIDDM: Type 2).

DESCRIPTION OF THE ART

[0002]Type 2 diabetes is a debilitating disease characterized by
high-circulating blood glucose, insulin and corticosteroid levels. The
incidence of type 2 diabetes is high and rising and is becoming a leading
cause of mortality, morbidity and healthcare expenditure throughout the
world (Amos et al., Diabetic Med. 14:S1-85, 1997). The causes of type 2
diabetes are not well understood. It is thought that both resistance of
target tissues to the action of insulin and decreased insulin secretion
("β-cell failure") occur. Major insulin-responsive tissues for
glucose homeostasis are liver, in which insulin stimulates glycogen
synthesis and inhibits gluconeogenesis; muscle, in which insulin
stimulates glucose uptake and glycogen and inhibits lipolysis. Thus, as a
consequence of the diabetic condition, there are elevated levels of
glucose in the blood, and prolonged high blood sugar that is indicative
of a condition which will cause blood vessel and nerve damage.

[0003]Currently, there are various pharmacological approaches for the
treatment of type 2 diabetes (Scheen et al., Diabetes Care,
22(9):1568-1577, 1999). One such approach is the use of
thiazolidinediones (TZDs), which represent a new class of oral
antidiabetic drugs that improve metabolic control in patients with type 2
diabetes. Their glucose-lowering effect is mediated through the
improvement of insulin sensitivity. They reduce insulin resistance in
adipose tissue, muscle and liver (Oakes et al., Metabolism 46:935-942,
(1997); Young et al. Diabetes 44:1087-1092, (1995); Oakes et al.,
Diabetes 43:1203-1210, (1994); Smith et al., Diabetes Obes Metab
2:363-372 (2000)). In addition, free fatty acid (FFA) levels were lowered
and there was a marked reduction in triglycerides.

[0005]FGF-21 has been reported to be preferentially expressed in the liver
(Nishimura et al., Biochimica et Biophysica Acta, 1492:203-206, (2000);
WO01/36640; and WO01/18172) and recently, has been shown to stimulate
glucose-uptake in mouse 3T3-L1 adipocytes after prolonged treatment, in
the presence and absence of insulin, and to decrease fed and fasting
blood glucose, triglycerides, and glucagon levels in ob/ob and db/db mice
in a dose-dependant manner, thus, providing the basis for the use of
FGF-21 as a therapy for treating diabetes and obesity (WO03/011213).

[0007]Accordingly, there is a need for an improved therapy of type 2
diabetes that has fewer adverse effects than the available pharmaceutical
approaches utilizing TZDs. The present invention provides a combination
therapy of FGF-21 with a TZD resulting in a synergistic effect that
enhances insulin sensitivity in peripheral tissues, stimulates glucose
uptake and has fewer adverse effects than treatment regimens for type 2
diabetes using TZDs alone or in combination with other agents.

SUMMARY OF THE INVENTION

[0008]The present invention provides a method for treating a mammal
exhibiting type 2 diabetes or metabolic syndrome comprising:
administering to said mammal a therapeutically effective amount of FGF-21
or an FGF-21 compound in combination with a thiazolidinedione sufficient
to achieve in said mammal at least one of the following modifications:
reduction in triglycerides, decrease in insulin resistance, reduction of
hyperinsulinemia, increase in glucose tolerance, or reduction of
hyperglycemia.

DETAILED DESCRIPTION OF THE INVENTION

[0009]FGF-21 is a 208 amino acid polypeptide containing a 27 amino acid
leader sequence. Human FGF-21 is highly identical to mouse FGF-21
(˜79% amino acid identity) and rat FGF-21 (˜80% amino acid
identity). Human FGF-21 is the preferred polypeptide of the present
invention but it is recognized that one with skill in the art could
readily use analogs, muteins, or derivatives of human FGF-21 or an
alternative mammalian FGF-21 polypeptide sequence for the uses described
herein.

[0012]The FGF-21 useful in the methods of the present invention is
preferably human FGF-21. Additionally, the methods of the present
invention include the use of FGF-21 analogs, FGF-21 muteins, and FGF-21
derivatives hereinafter collectively known as FGF-21 compounds. FGF-21
compounds have sufficient homology to FGF-21 such that the compound has
the ability to bind to the FGF-21 receptor and initiate a signal
transduction pathway resulting in glucose uptake stimulation or other
physiological effects as described herein. For example, FGF-21 compounds
can be tested for glucose uptake activity using a cell-based assay such
as that described in Example 1.

[0013]A human FGF-21 mutein is defined as comprising human FGF-21 in which
at least one amino acid of the wild-type mature protein has been
substituted by another amino acid. Examples of FGF-21 muteins are
described in U.S. patent application 60/528,582 herein incorporated by
reference. Generally speaking, a mutein possesses some modified property,
structural or functional, of the wild-type protein. For example, the
mutein may have enhanced or improved physical stability in concentrated
solutions (e.g., less hydrophobic mediated aggregation), while
maintaining a favorable bioactivity profile. The mutein may possess
increased compatibility with pharmaceutical preservatives (e.g.,
m-cresol, phenol, benzyl alcohol), thus enabling the preparation of a
preserved pharmaceutical formulation that maintains the physiochemical
properties and biological activity of the protein during storage.
Accordingly, muteins with enhanced pharmaceutical stability when compared
to wild-type FGF-21, have improved physical stability in concentrated
solutions under both physiological and preserved pharmaceutical
formulation conditions, while maintaining biological potency. As used
herein, these terms are not limiting, it being entirely possible that a
given mutein has one or more modified properties of the wild-type
protein.

[0014]An FGF-21 compound also includes a "FGF-21 derivative" which is
defined as a molecule having the amino acid sequence of FGF-21 or an
FGF-21 analog, but additionally having a chemical modification of one or
more of its amino acid side groups, α-carbon atoms, terminal amino
group, or terminal carboxylic acid group. A chemical modification
includes, but is not limited to, adding chemical moieties, creating new
bonds, and removing chemical moieties.

[0015]Modifications at amino acid side groups include, without limitation,
acylation of lysine εamino groups, N-alkylation of arginine,
histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid
groups, and deamidation of glutamine or asparagine. Modifications of the
terminal amino group include, without limitation, the des-amino, N-lower
alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the
terminal carboxy group include, without limitation, the amide, lower
alkyl amide, dialkyl amide, and lower alkyl ester modifications.
Furthermore, one or more side groups, or terminal groups, may be
protected by protective groups known to the ordinarily-skilled protein
chemist. The α-carbon of an amino acid may be mono- or
dimethylated.

[0016]Type 2 diabetes is characterized by excess glucose production in
spite of the availability of insulin, and circulating glucose levels
remain excessively high as a result of inadequate glucose clearance.

[0017]Glucose intolerance can be defined as an exceptional sensitivity to
glucose.

[0018]Hyperglycemia is defined as an excess of sugar (glucose) in the
blood.

[0020]Hyperinsulinemia is defined as a higher-than-normal level of insulin
in the blood.

[0021]Insulin resistance is defined as a state in which a normal amount of
insulin produces a subnormal biologic response.

[0022]Metabolic syndrome can be defined as a cluster of at least three of
the following signs: abdominal fat--in most men, a 40-inch waist or
greater; high blood sugar--at least 110 milligrams per deciliter (mg/dl)
after fasting; high triglycerides--at least 150 mg/dL in the bloodstream;
low HDL--less than 40 mg/dl; and, blood pressure of 130/85 or higher.

[0023]The FGF-21 administered according to this invention may be generated
and/or isolated by any means known in the art such as described in
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, NY (1989).

[0024]Various methods of protein purification may be employed and such
methods are known in the art and described, for example, in Deutscher,
Methods in Enzymology 182: 83-9 (1990) and Scopes, Protein Purification:
Principles and Practice, Springer-Verlag, NY (1982). The purification
step(s) selected will depend, for example, on the nature of the
production process used for FGF-21.

[0025]TZDs are formulated as described in the art. For example, the TZDs
rosiglitazone (Avandia®) and pioglitazone (Actos®) are currently
used as pharmaceutical compositions administered alone or in combination
with metformin or sulfonylureas for the treatment of type 2 diabetes.

[0026]The pharmaceutical compositions of the TZDs of the present invention
may be administered by any means that achieve the generally intended
purpose: to treat type 2 diabetes or metabolic syndrome. Preferably, the
TZD is administered orally.

[0027]FGF-21 utilized in combination with a TZD may be formulated
according to known methods to prepare pharmaceutically useful
compositions. A desired formulation would be one that is a stable
lyophilized product that is reconstituted with an appropriate diluent or
an aqueous solution of high purity with optional pharmaceutically
acceptable carriers, preservatives, excipients or stabilizers
[Remington's Pharmaceutical Sciences 16th edition (1980)]. The FGF-21 of
the present invention may be combined with a pharmaceutically acceptable
buffer, and the pH adjusted to provide acceptable stability, and a pH
acceptable for administration.

[0028]For parenteral administration FGF-21 is formulated generally, in a
unit dosage injectable form (solution, suspension, or emulsion), with a
pharmaceutically acceptable carrier, i.e. Preferably, one or more
pharmaceutically acceptable anti-microbial agents may be added. Phenol,
m-cresol, and benzyl alcohol are preferred pharmaceutically acceptable
anti-microbial agents.

[0029]Optionally, one or more pharmaceutically acceptable salts may be
added to adjust the ionic strength or tonicity. One or more excipients
may be added to further adjust the isotonicity of the formulation.
Glycerin, sodium chloride, and mannitol are examples of an isotonicity
adjusting excipient.

[0030]"Pharmaceutically acceptable" means suitable for administration to a
human. A pharmaceutically acceptable formulation does not contain toxic
elements, undesirable contaminants or the like, and does not interfere
with the activity of the active compounds therein.

[0031]If subcutaneous or an alternative type of administration is used,
the FGF-21 compounds may be derivatized or formulated such that they have
a protracted profile of action.

[0032]A "therapeutically effective amount" of FGF-21 or an FGF-21 compound
is the quantity that results in a desired effect without causing
unacceptable side-effects when administered to a subject. A desired
effect can include an amelioration of symptoms associated with the
disease or condition, a delay in the onset of symptoms associated with
the disease or condition, and increased longevity compared with the
absence of treatment. In particular, the desired effect is a reduction in
blood glucose levels or triglerceride levels associated with type 2
diabetes or metabolic syndrome.

[0033]The phrase "in combination with" refers to the administration of
FGF-21 with a TZD either simultaneously, sequentially or a combination
thereof. Preferably, the TZD is administered orally and the FGF-21 is
administered parenterally. The combination therapy of FGF-21 with a TZD
results in a synergistic effect with enhanced efficacy in the treatment
of type 2 diabetes. The synergy also results in a reduction of the dosage
of the agents used in combination therapy resulting in reduced side
effects such as weight gain, liver toxicity, upper respiratory tract
infection, headache, back pain, hyperglycemia, fatigue, sinusitis,
diarrhea, hypoglycemia, mild to moderate edema, and anemia.

[0034]The TZD utilized and the appropriate dose level is understood and
appreciated in the art. A skilled artisan recognizes the appropriate dose
level to use for each TZD to achieve a pharmaceutically effective amount
for treating type 2 diabetes. TZDs agents suitable for use under the
present invention include, but are not limited to, clinically recognized
and commercially available agents such as rosiglitazone, pioglitazone,
and troglitazone (Hauner, H., Diabetes Metab Res Rev 18:S10-S15 (2002)).
Typically, the amount of rosiglitazone administered for the treatment of
type 2 diabetes is from 4 mg to 8 mg per day and the amount of
pioglitazone administered for the treatment of type 2 diabetes is from 15
mg to 45 mg per day.

[0035]The pharmaceutical compositions of the FGF-21 of the present
invention may be administered by any means that achieve the generally
intended purpose: to treat type 2 diabetes or metabolic syndrome. For
example, administration may be by oral, ocular, optical, rectal,
parenteral, intravaginal, topical (as by powders, ointments, drops, or
transdermal patch), bucal, as an oral or nasal spray, or as ocular or
intraotic drops. The term "parenteral" as used herein refers to modes of
administration that include intravenous, intramuscular, intraperitoneal,
intrastemal, subcutaneous, and intraarticular injection and infusion. The
dosage administered will be dependent upon the age, health, and weight of
the recipient, kind of concurrent treatment, if any, frequency of
treatment, and the nature of the effect desired. Compositions within the
scope of the invention include all compositions wherein FGF-21 is present
in an amount that is effective to achieve the desired medical effect for
treatment type 2 diabetes or metabolic syndrome. While individual needs
may vary from one patient to another, the determination of the optimal
ranges of effective amounts of all of the components is within the
ability of the clinician of ordinary skill.

[0036]Those skilled in the art can readily optimize pharmaceutically
effective dosages and administration regimens for therapeutic
compositions comprising FGF-21, as determined by good medical practice
and the clinical condition of the individual patient. A typical dose
range for FGF-21 will range from about 0.01 mg per day to about 1000 mg
per day for an adult. Preferably, the dosage ranges from about 0.1 mg per
day to about 100 mg per day, more preferably from about 1.0 mg/day to
about 10 mg/day. Most preferably, the dosage is about 1-5 mg/day. The
appropriate dose of FGF-21 administered will result in lowering blood
glucose levels and increasing energy expenditure by faster and more
efficient glucose utilization, and thus is useful for treating type 2
diabetes or metabolic syndrome.

[0037]Alternatively, FGF-21 is administered twice weekly at a dose range
from about 0.01 mg per dose to about 1000 mg per dose for an adult.
Preferably, the dosage ranges from about 0.1 mg per dose to about 100 mg
per dose, more preferably from about 1.0 mg per dose to about 10 mg per
day. Most preferably, the dosage is about 1-5 mg per dose.

[0038]In another alternative, FGF-21 is administered once weekly at a dose
range from about 0.01 mg per dose to about 1000 mg per dose for an adult.
Preferably, the dosage ranges from about 0.1 mg per dose to about 100 mg
per dose, more preferably from about 1.0 mg per dose to about 10 mg per
dose. Most preferably, the dosage is about 1-5 mg per dose.

[0039]FGF-21 administered either daily, twice weekly or once weekly,
combined with a TZD such as rosiglitazone or pioglitazone, has a
synergistic effect in the treatment of type 2 diabetes that improves the
efficacy of the TZD alone. Thus, this combination therapy reduces the
therapeutic dose of the TZD required for therapeutic treatment of type 2
diabetes thereby minimizing the side effects typically observed with TZD
therapy. For example the amount of TZD administered in combination with
FGF-21 is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or to about
80% of the typical dose of TZD utilized in the treatment of type 2
diabetes.

[0040]In another aspect of the present invention, FGF-21 in combination
with a TZD for use as a medicament for the treatment of type 2 diabetes
or metabolic syndrome is contemplated.

[0041]Having now described the present invention in detail, the same will
be more clearly understood by reference to the following examples, which
are included herewith for purposes of illustration only and are not
intended to be limiting of the invention.

[0042]All patents and publications referred to herein are expressly
incorporated by reference.

Preparation 1

Expression and Purification of FGF-21 in E. coli

[0043]The bacterial expression vector pET30a is used for bacterial
expression in this example. (Novagen, Inc., Madison, Wis.)). pET30a
encodes kanamycin antibiotic resistance gene and contains a bacterial
origin of replication ("ori"), a strong T7 phage-IPTG inducible promoter,
a ribosome binding site ("RBS"), and suitable MCS with a number of unique
restriction endonuclease cleavage sites. Conveniently for purification
purpose, the vector can encode His- and S-tags for N-terminal peptide
fusions, as well as, a C-terminal His-tag fusion. However, for purposes
of the present invention, the cDNA encoding FGF-21 is inserted between
restriction sites NdeI and BamHI, respectively, and the resulting
construct does not take advantage of either of the described tags.

[0044]The nucleic acid sequence encoding FGF-2, lacking the leader
sequence but substituted with a methionine residue, is amplified from a
cDNA clone using PCR oligonucleotide primers, which anneal to the 5' and
3' ends of the open reading frame. Additional nucleotides, containing
recognition sites for restriction enzymes NdeI and BamHI, are added to
the 5' and 3' sequences, respectively.

[0045]For cloning, the 5' forward and 3' reverse PCR primers have
nucleotides corresponding or complementary to a portion of the coding
sequence of FGF-21-encoding nucleic acid according to methods known in
the art. One of ordinary skill in the art would appreciate that the point
in a polynucleotide sequence where primers begin can be varied.

[0046]The amplified nucleic acid fragments and the vector pET30a are
digested with NdeI and BamHI restriction enzymes and the purified
digested DNA fragments are then ligated together. Insertion of FGF-21
mutein-encoding DNA into the restricted pET30a vector places the FGF-21
mutein polypeptide coding region including its associated stop codon
downstream from the IPTG-inducible promoter and in-frame with an
initiating ATG codon. The associated stop codon, TAG, prevents
translation of the six-histidine codons downstream of the insertion
point,

[0047]The ligation mixture is transformed into competent E. coli cells
using standard procedures such as those described in Current Protocols in
Molecular Biology (John Wiley & Sons, Inc.).

[0048]Transformation reactions are plated on LB/Kanamycin plates and after
an overnight growth transformants are picked for plasmid preparations or
lysed in situ for screening by PCR. Positive recombinant plasmids,
containing desired FGF-21 variant inserts, are identified by restriction
analysis followed by DNA sequence analysis. Those plasmids are
subsequently used to transform expression strains and protein production.

[0049]E. coli strains BL21(DE3), BL21(DE3)STAR or BL21(DE3) RP, are used
for expressing FGF-21. These strains, which are only some of many that
are suitable for expressing FGF-21, are available commercially from
Novagen, Inc., Invitrogen and Stratagen, respectively. Transformants are
identified by their ability to grow on LB plates in the presence of
kanamycin.

[0050]Clones containing the desired constructs are grown overnight (o/n)
in liquid culture in LB media supplemented with kanamycin (30 μg/ml).
The o/n culture is used to inoculate a large culture, at a dilution of
approximately 1:25 to 1:250. The cells are grown to an optical density of
0.6 ("OD600") at 600 nm. Isopropyl-b-D-thiogalactopyranoside ("IPTG") is
then added to a final concentration of 1 mM to induce transcription from
the lac repressor sensitive promoter, by inactivating the lacI repressor.
Cells subsequently are incubated further for 3 to 12hours. Cells are then
harvested by centrifugation, pellets washed with 50 mM Tris buffer, pH
8.0 and stored at -20° C. until purification. FGF-21 is expressed
in the insoluble fraction i.e inclusion bodies (or granules) of E. coli.
Although the expression level may a typically observed level for FGF-21
protein is 50 mg/L. The subsequent purification process starts with
solubilization of the granules and refolding of the variants followed by
four chromatographic steps.

[0051]To purify FGF-21 from E coli, the granules are solubilized in 50 mM
Tris, pH 9.0, 7M Urea and 1 mM DTT through a pH ramp to pH 11.0, at room
temperature for 1 hour with stirring. The protein is then captured on a
Q-Sepharose column using the same buffer described above, and eluted with
a linear gradient of 0-400 mM NaCl. The Q-Sepharose pool is then treated
with 10 mM DTT, for two hours, at RT, to reduce all disulfide bonds. The
pool is then diluted 10-fold so that the buffer concentration is as
follows: 50 mM Tris, pH 9.0, 7 M Urea, 10 mM Cysteine, 1 mM DTT with a
protein concentration of approximately 250-500 μg/ml. After another
two-hour incubation under reducing conditions at RT, to obtain the
protein in a free disulfide form, the pool is then dialyzed into 20 mM
glycine, pH 9.0 for approximately 48 hours so that the correct disulfide
bonds can be formed.

[0052]Reversed-phase HPLC chromatography, on a Vydac C18 column and 0.1%
TFA/0-50% CH3CN as a mobile phase is used as an initial purification
step. This column is used to concentrate FGF-21 and removes contaminating
endotoxin.

[0053]The next purification step is size exclusion chromatography on a
Superdex 35/600 column performed in 1×PBS buffer, pH7.4. At this
step FGF-21 is ˜95% pure. The last step involves MonoQ
chromatography in 50 mM Tris, pH 8.0 and elution with a linear gradient
of 0-300 mM NaCl, which usually yields >97% pure protein.

Preparation 2

Expression and Purification of FGF-21 in HEK293EBNA Cells

[0054]Alternatively, FGF-21 is produced in a mammalian cell expression
system such as HEK293EBNA cells (EdgeBiosystems, Gaiethersburg, Md.).
FGF-21 is subcloned in the proprietary expression vector representing a
modification of commercially available pEAK10, between NheI and XbaI
restriction sites in the MCS. The cDNA sequence encoding mature FGF-21 is
fused in frame with the Igκ leader sequence to enhance secretion of
the desired product in the tissue culture media. The expression is driven
by the strong viral CMV promoter. HEK293EBNA cells are transiently
transfected using a standard transfection reagent such as Fugene (Roche
Diagnostics, Indianapolis, Ind.) and the appropriate amount of
recombinant plasmid, either as a monolayer or suspension culture, at the
adequate cell density. Cells are incubated at 37° C. and 5%
CO2, in serum free media, and collections are made every day for 5
days. Typically the expression level in the HEK239EBNA suspension culture
is ˜30 mg/L. The expression of human FGF-21 in mammalian cells
yields the natural N-terminus sequence of HPIP, i.e. without a methionine
residue at the N-terminus.

Preparation 3

Expression and Purification of FGF-21 in Yeast

[0055]Yet another expression system for production of FGF-21 is yeast,
such as Pichia pastoris, Pichia methanolica or Saccharomyces cerevisiae.
For production in Pichia pastoris, a commercially available system
(Invitrogen, Carlsbad, Calif.) uses vectors with the powerful AOX1
(alcohol oxidase) promoters to drive high-level expression of recombinant
proteins. Alternatively, vectors that use the promoter from the GAP gene
(glyceraldehyde-3-phosphate dehydrogenase) are available for high level
constitutive expression. The multi-copy Pichia expression vectors allow
one to obtain strains with multiple copies of the gene of interest
integrated into the genome. Increasing the number of copies of the gene
of interest in a recombinant Pichia strain can increase protein
expression levels.

[0057]Glucose Transport Assay--FGF-21 is added to the differentiated
3T3-L1 cells in 96 well plates at 0, 0.016, 0.08, 0.4, 2, 10, or 50.0 nM,
and rosiglitazone is added to a final concentration of 1 μM, Table 1.
For comparison, rosiglitazone alone is added at the concentrations
indicated in Table 2 or in combination with FGF-21 at 1 μg/ml. The
plates are incubated at 37° C. for 72 hours.

[0059]The in vitro potency of FGF-21 alone or in combination with
rosiglitazone is indicated in Table 1. FGF-21 alone has an ED50 of
1.7 nM whereas FGF-21 in combination with rosiglitazone demonstrates a
synergistic effect and has an ED50 of 0.7 nM.

[0060]The in vitro potency of rosiglitazone alone or in combination with
FGF-2 1 at 1 μg/ml is indicated in Table 2. Rosiglitazone alone has no
effect on glucose uptake in 3T3 cells whereas rosiglitazone in
combination with FGF-21 has an ED50 of 0.007 μM.